Method for the treatment and prevention of ocular disorders

ABSTRACT

Provided herein are methods for treatment of ocular diseases. The methods comprise administering a compound of general Formula (I): wherein R 1 , R 2 , R 3 , R 4  and Ak are as defined in the specification.

This application claims priority to U.S. provisional application Ser.No. 60/802,208 filed May 19, 2007, entitled “METHODS FOR THE TREATMENTAND PREVENTION OF OCULAR DISORDERS” to David Houck. The disclosure ofthe above referenced application is incorporated by reference herein.

1 FIELD OF THE INVENTION

Provided herein are methods of using cyclosporin compounds andcompositions in treatment or prevention of ocular diseases and disorderssuch as aqueous deficient dry-eye state, uveitis and phacoanaphylacticendophthalmitis. In certain aspects, the compounds for use in themethods provided herein are 3-alkylaminoalkyl, 3-dialkylaminoalkyl or3-heterocyclylalkyl substituted cyclosporin compounds. In certainembodiments, the methods comprise administering to a subject in needthereof an amount of the compound provided herein effective to treat orprevent the ocular diseases and disorders such as aqueous deficientdry-eye state, uveitis and phacoanaphylactic endophthalmitis.

2 BACKGROUND

Dry-eye state may occur in a wide range of individuals, although it ismore frequently seen in women, the elderly, and those with connectivetissue disorders (e.g., rheumatoid arthritis, Sjögren's syndrome).Patients with dry eye commonly have complaints of ocular irritation ordiscomfort. As the name implies, dryness is the most frequently citedproblem; patients may further report itching, burning, or a“sandy/gritty” foreign body sensation. Symptoms may be exacerbated bypoor air quality, low humidity or extreme heat, and tend to be moreprominent later in the day. Occasionally, patients report excesslacrimation, or epiphora, in association with the discomfort.

Uveitis, the inflammation of the uvea, is responsible for about 10% ofthe visual impairment in the United States. The uveal tract of the eyeconsists of the iris, ciliary body, and choroid. Inflammation of theoverlying retina, called retinitis, or of the optic nerve, called opticneuritis, may occur with or without accompanying uveitis.

Uveitis is most commonly classified anatomically as anterior,intermediate, posterior, or diffuse. Anterior uveitis is localizedprimarily to the anterior segment of the eye and includes iritis andiridocyclitis. Intermediate uveitis, also called peripheral uveitis, iscentered in the area immediately behind the iris and lens in the regionof the ciliary body and pars plana, hence the alternate terms “cyclitis”and “pars planitis” are also used. Posterior uveitis signifies any of anumber of forms of retinitis, choroiditis, or optic neuritis. Diffuseuveitis implies inflammation involving all parts of the eye, includinganterior, intermediate, and posterior structures.

Phacoanaphylactic endophthalmitis is a human autoimmune disease. It isan inflammatory ocular condition secondary to rupture of the lenscapsule, either traumatically or iatrogenically is also referred to aslens induced uveitis. Phacoanaphylaxis is a severe form of uveitis inwhich the lens is the causative antigen. The lens proteins are normallysecluded by the lens capsule since before birth. When these proteins arereleased into the eye by injury or surgery or occasionally duringcataract development, they can become intensely antigenic and incite anautoimmune response. If the response is moderate it is seen as a chronicuveitis. If it is very fast in progression the eye becomes severelyinflamed in all segments. This latter response is namedphacoanaphylaxis.

There is a continuing need to develop new and effective compounds totreat ocular diseases and disorders, such as dry eye, uveitis andphacoanaphylactic endophthalmitis.

Cyclosporins are a group of nonpolar cyclic oligopeptides withimmunosuppressant, anti-inflammatory, and anti-parasitic properties.Cyclosporin A is a cyclosporin which is marketed in a topical ophthalmicemulsion formulation for the treatment of dry eye under the tradenameRestasis. The insolubility of cyclosporins in water is an ongoingproblem in the formulation of these compounds. In one aspect, thepresent invention seeks to provide cyclosporin derivatives havingimproved water solubility properties in comparison with cyclosporin Awhile maintaining useful properties for treating ocular diseases.

3 SUMMARY

Provided herein are methods for treating or preventing ocular diseasescomprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound of general formula (I):

wherein:

-   Ak is alkylene;-   R¹ is —NR⁵R⁶, in which R⁵ and R⁶ are each independently hydrogen or    straight- or branched-chain alkyl comprising from one to six carbon    atoms; or R⁵ and R⁶, together with the nitrogen atom to which they    are attached, form a saturated or unsaturated heterocyclic ring    containing from four to six ring atoms, which ring may optionally    contain another heteroatom selected from the group consisting of    nitrogen, oxygen and sulfur and may be optionally substituted by one    to four groups which may be the same or different selected from    alkyl, hydroxyl, amino, N-alkylamino and N,N-dialkylamino;-   R² is isobutyl;-   R³ is (E)-2-butenyl-1 or n-butyl;-   R⁴ is ethyl, 1-hydroxyethyl, isopropyl or n-propyl;    or a pharmaceutically acceptable salt or solvate thereof.

4 DETAILED DESCRIPTION

Provided are methods of treating, preventing or ameliorating oculardiseases and disorders in a subject in need thereof In one embodimentthe present invention provides compounds of general formula (I) asdefined above or pharmaceutically acceptable salts or solvates thereof,in the manufacture of a medicament for the treatment or prevention ofocular diseases. The methods and compositions are described in detail inthe sections below.

4.1 Definitions

When referring to the compounds and complexes provided herein, thefollowing terms have the following meanings unless indicated otherwise.

“Cyclosporin” refers to any cyclosporin compound known to those of skillin the art, or a derivative thereof. See, e.g., Ruegger et al., 1976,Helv. Chim. Acta. 59:1075-92; Borel et al., 1977, Immunology 32:1017-25;the contents of which are hereby incorporated by reference in theirentireties. Exemplary compounds for use in the methods provided hereinare cyclosporin derivatives. Unless noted otherwise, a cyclosporindescribed herein is a cyclosporin A, and a cyclosporin derivativedescribed herein is a derivative of cyclosporin A.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups,particularly having up to about 11 carbon atoms, more particularly as alower alkyl, from 1 to 8 carbon atoms and still more particularly, from1 to 6 carbon atoms. The hydrocarbon chain may be eitherstraight-chained or branched. This term is exemplified by groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl,n-hexyl, n-octyl, tert-octyl and the like. The term “lower alkyl” refersto alkyl groups having 1 to 6 carbon atoms.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupsparticularly having up to about 11 carbon atoms and more particularly 1to 6 carbon atoms which can be straight-chained or branched. This termis exemplified by groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—),the propylene isomers (e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Amino” refers to the radical —NH_(2.)

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g. heteroalkyl, aryl, e.g. heteroaryl, and the like having from1 to 5, and especially from 1 to 3 heteroatoms.

“Heterocycle” or “heterocyclic ring” refers to any heterocycle known tothose of skill in the art. As used herein, a heterocycle can be aheteroaryl group or a cycloheteroalkyl group, as will be recognized bythose of skill in the art. In certain embodiments, heterocyclyl refersto a 4, 5, or 6 membered saturated heterocyclic ring containing one ormore heteroatoms in the ring.

“Dialkylamino” means a radical —NRR′ where R and R′ independentlyrepresent an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substitutedcycloheteroalkyl, heteroaryl, or substituted heteroaryl group as definedherein.

“Halogen” or “halo” refers to chloro, bromo, fluoro or iodo.

“Pharmaceutically acceptable salt” refers to any salt of a compoundprovided herein which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art and include. Such salts include: (1) acid addition saltsformed with organic or inorganic acids such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic,trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2)salts formed when an acidic proton present in the parent compound either(a) is replaced by a metal ion, e.g., an alkali metal ion, an alkalineearth ion or an aluminum ion, or alkali metal or alkaline earth metalhydroxides, such as sodium, potassium, calcium, magnesium, aluminum,lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with anorganic base, such as aliphatic, alicyclic, or aromatic organic amines,such as ammonia, methylamine, dimethylamine, diethylamine, picoline,ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylene-diamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like.

Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium and the like, and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrohalides, e.g. hydrochloride andhydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate,trifluoroacetate, trichloroacetate, propionate, hexanoate,cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate,cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate,benzenesulfonate (besylate), 4-chlorobenzenesulfonate,2-naphthalenesulfonate, 4-toluenesulfonate, camphorate,camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate,glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate,lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,salicylate, stearate, cyclohexylsulfamate, quinate, muconate and thelike.

It is to be understood that where reference is made in the presentspecification to the compounds of general formula, such reference isintended to include also the solvates, hydrates or salts withpharmaceutically acceptable acids or bases of compounds of generalFormula (I) where appropriate.

“Sarcosine” refers to N-methyl glycine (CH₃NHCH₂CO₂H).

“Solvate” refers to a compound provided herein or a salt thereof thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

It is to be understood that compounds having the same molecular formulabut differing in the nature or sequence of bonding of their atoms or inthe arrangement of their atoms in space are termed “isomers.” Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers.”

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, when it is bonded to four different groups, a pairof enantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is designated (R) or(S) according to the rules of Calm and Prelog (Calm et al., 1966, Angew.Chem. 78:413-447, Angew. Chem., Int. Ed. Engl. 5:385-414 (errata: Angew.Chem., Int. Ed. Engl. 5:511); Prelog and Helmchen, 1982, Angew. Chem.94:614-631, Angew. Chem. Internat. Ed. Eng. 21:567-583; Mata and Lobo,1993, Tetrahedron: Asymmetry 4:657-668) or can be characterized by themanner in which the molecule rotates the plane of polarized light and isdesignated dextrorotatory or levorotatory (i.e., as (+)- or (−)-isomers,respectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of enantiomers is called a “racemic mixture.”

In certain embodiments, the compounds for use in the methods providedherein may possess one or more asymmetric centers; such compounds cantherefore be produced as the individual (R)- or (S)-enantiomer or as amixture thereof. Unless indicated otherwise, for example by designationof stereochemistry at any position of a formula, the description ornaming of a particular compound in the specification and claims isintended to include both individual enantiomers and mixtures, racemic orotherwise, thereof. Methods for determination of stereochemistry andseparation of stereoisomers are well-known in the art. In particularembodiments, provided herein are the stereoisomers of the compoundsdepicted herein upon treatment with base.

In certain embodiments, the compounds for use in the methods providedherein are “stereochemically pure.” A stereochemically pure compound orhas a level of stereochemical purity that would be recognized as “pure”by those of skill in the art. Of course, this level of purity will beless than 100%. In certain embodiments, “stereochemically pure”designates a compound that is substantially free of alternate isomers.In particular embodiments, the compound is 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% free of other isomers.

As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as acynomolgous monkey, a chimpanzee and a human), including a human. Inanother embodiment, the subject is a farm animal (e.g., a horse, a cow,a pig, etc.) or a pet (e.g., a dog or a cat). In certain embodiments,the subject is a human.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disorder or one or more symptoms thereof. In certain embodiments,the term “therapeutic agent” refers to a compound provided herein. Incertain other embodiments, the term “therapeutic agent” refers does notrefer to a compound provided herein. A therapeutic agent is an agentwhich is known to be useful for, or has been or is currently being usedfor the treatment or prevention of a disorder or one or more symptomsthereof.

“Therapeutically effective amount” means an amount of a compound orcomplex or composition that, when administered to a subject for treatinga disease, is sufficient to effect such treatment for the disease. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the disease and its severity, and the age, weight, etc.,of the subject to be treated.

“Treating” or “treatment” of any disease or disorder refers, in oneembodiment, to ameliorating a disease or disorder that exists in asubject. In another embodiment, “treating” or “treatment” refers toameliorating at least one physical parameter, which may be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”refers to modulating the disease or disorder, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” refers to delaying the onset ofthe disease or disorder.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisorder or one or more symptoms thereof. In certain embodiments, theterm “prophylactic agent” refers to a compound provided herein. Incertain other embodiments, the term “prophylactic agent” does not refera compound provided herein. In certain embodiments, a prophylactic agentis an agent which is known to be useful for, or has been or is currentlybeing used to prevent or impede the onset, development, progressionand/or severity of a disorder.

As used herein, the terms “prevent,” “ preventing” and “prevention”refer to the prevention of the recurrence, onset, or development of oneor more symptoms of a disorder in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or the administration of a combination of therapies (e.g., a combinationof prophylactic or therapeutic agents).

As used herein, the phrase “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention of the development, recurrence or onset ofone or more symptoms associated with a disorder (, or to enhance orimprove the prophylactic effect(s) of another therapy (e.g., anotherprophylactic agent).

As used herein, the term “in combination” refers to the use of more thanone therapy (e.g., one or more prophylactic and/or therapeutic agents).The use of the term “in combination” does not restrict the order inwhich therapies (e.g., prophylactic and/or therapeutic agents) areadministered to a subject with a disorder. A first therapy (e.g., aprophylactic or therapeutic agent such as a compound provided herein)can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of a second therapy (e.g., aprophylactic or therapeutic agent) to a subject with a disorder.

As used herein, the term “synergistic” refers to a combination of acompound provided herein and another therapy (e.g., a prophylactic ortherapeutic agent) which has been or is currently being used to prevent,manage or treat a disorder, which is more effective than the additiveeffects of the therapies. A synergistic effect of a combination oftherapies (e.g., a combination of prophylactic or therapeutic agents)permits the use of lower dosages of one or more of the therapies and/orless frequent administration of said therapies to a subject with adisorder. The ability to utilize lower dosages of a therapy (e.g., aprophylactic or therapeutic agent), and/or to administer said therapyless frequently, reduces the toxicity associated with the administrationof said therapy to a subject without reducing the efficacy of saidtherapy in the prevention or treatment of a disorder). In addition, asynergistic effect can result in improved efficacy of agents in theprevention or treatment of a disorder. Finally, a synergistic effect ofa combination of therapies (e.g., a combination of prophylactic ortherapeutic agents) may avoid or reduce adverse or unwanted side effectsassociated with the use of either therapy alone.

4.2 Embodiments of the Invention

Provided herein are methods of treating, preventing or amelioratingocular diseases and disorders such as aqueous deficient dry-eye state,uveitis and phacoanaphylactic endophthalmitis comprising administeringin a subject in need thereof a compound of formula I.

Without wishing to be limited to any particular theory of operation, itis believed that, in certain embodiments, the compounds act to enhanceor restore lacrimal gland tearing in providing the desired therapeuticeffect.

4.2.1 Compounds for Use in the Methods

Unless noted otherwise, the term “cyclosporin” as used herein refers tothe compound cyclosporin A as known to those of skill in the art. See,e.g., Ruegger et al., 1976, Helv. Chim. Acta. 59:1075-92; Borel et al.,1977, Immunology 32:1017-25; the contents of which are herebyincorporated by reference in their entireties. The term “cyclosporincompound” refers to any cyclosporin compound with activity againstocular disease described herein, whether the compound is natural,synthetic or semi-synthetic.

In certain embodiments, Ak is —(CH₂)_(p)—, where p is 1, 2, 3 or 4;and/or R¹ is —NR⁵R⁶ and R⁵ and R⁶ are selected as follows a) R⁵ and R⁶are each independently straight- or branched-chain alkyl comprising fromone to six carbon atoms or b) R⁵ and R⁶, together with the nitrogen atomto which they are attached, form a saturated or unsaturated heterocyclicring containing from five to six ring atoms, which ring may optionallycontain another heteroatom selected from the group consisting ofnitrogen, oxygen and sulfur. In certain embodiments, Ak is —CH₂CH₂—.

In certain embodiments R¹ is —NR⁵R⁶, in which R⁵ and R⁶ are eachindependently hydrogen or straight- or branched-chain alkyl comprisingfrom one to six carbon atoms; or R⁵ and R⁶, together with the nitrogenatom to which they are attached, form a saturated or unsaturatedheterocyclic ring containing from five to six ring atoms, which ring mayoptionally contain another heteroatom selected from the group consistingof nitrogen, oxygen and sulfur and may be optionally substituted by fromone to four alkyl groups which may be the same or different.

In certain embodiments, R⁵ and R⁶ are both methyl. In certainembodiments, R⁵ and R⁶, together with the nitrogen atom to which theyare attached, form a saturated heterocyclic ring containing six ringatoms, which ring may optionally contain another heteroatom selectedfrom the group consisting of nitrogen, oxygen and sulfur. In a furtherembodiment, R⁵ and R⁶, together with the nitrogen atom to which they areattached, form an azetidinyl ring. In a still further embodiment, R⁵ andR⁶, together with the nitrogen atom to which they are attached, form apyrrolidinyl ring. In certain embodiments, R⁵ and R⁶, together with thenitrogen atom to which they are attached, form a piperidinyl ring. Incertain embodiments, R⁵ and R⁶, together with the nitrogen atom to whichthey are attached, form an N-methylpiperazinyl ring. In certainembodiments, R⁵ and R⁶, together with the nitrogen atom to which theyare attached, form a morpholinyl ring. In certain embodiments, R⁵ andR⁶, together with the nitrogen atom to which they are attached, form a2,6-dimethylmorpholinyl ring.

In particular embodiments, the cyclosporin compound differs fromcyclosporin A at the third position, i.e. the N-methyl glycine position,known to those of skill in the art. In certain embodiments, thecyclosporin compound comprises a 3-alkylaminoalkyl,3-N,N-dialkylaminoalkyl or 3-heterocyclylalkyl group. The cyclosporincompound can further comprise other cyclosporin modifications known tothose of skill in the art.

In certain embodiments, the methods provided herein compriseadministering to the subject a therapeutically or prophylacticallyeffective amount of a cyclosporin compound of general Formula (I) abovewherein B, X, R¹, R² and R³ are as defined above, or a pharmaceuticallyacceptable salt or solvate thereof

In certain embodiments, R³ is (E)-2-butenyl-1. In certain embodiments R³is n-butyl.

In certain embodiments, R⁴ is ethyl.

In a further embodiment Ak is (CH₂)_(p) and p is two or three; R¹ is—NR⁵R⁶; R⁵ and R⁶, which may be the same or different, are hydrogen orC₁₋₃ alkyl; or R⁵ and R⁶, together with the nitrogen atom to which theyare attached, form a saturated or unsaturated heterocyclic ringcontaining from four to six ring atoms, which ring may optionallycontain another heteroatom selected from the group consisting ofnitrogen and oxygen and may be optionally substituted by one or twogroups which may be the same or different selected from methyl, hydroxyland dimethylamino; R² is isobutyl; R³ is (E)-2-butenyl-1; and R⁴ isethyl.

Among the compounds used in the methods provided herein, are thecyclosporin compounds listed below:

-   A 3-[2-(N,N-dimethylamino)ethoxy]cyclosporin.-   B 3-[2-(azetidin-1-yl)ethoxy]cyclosporin.-   C 3-[2-(pyrrolidin-1-yl)ethoxy]cyclosporin.-   D 3-[2-(piperidin-1-yl)ethoxy]cyclosporin.-   E 3-[2-(4-methylpiperazin-1-yl)ethoxy]cyclosporin.-   F 3-[2-(morpholin-4-yl)ethoxy]cyclosporin.-   G 3-[2-(2,6-dimethylmorpholin-4-yl)ethoxy]cyclosporin.-   H 3-[2-(3-hydroxypyrrolidin-1-yl)ethoxy]cyclosporin.-   I 3-{2-[(4-dimethylamino)piperidin-1-yl]ethoxy}cyclosporin.-   J 3-[2-(4-hydroxypiperidin-1-yl)ethoxy]cyclosporin.-   K 3-[2-(imidazol-1-yl)ethoxy]cyclosporin.-   L 3-[2-(N-methylamino)ethoxy]cyclosporin.-   M 3-[2-(N-isopropyl-N-methylamino)ethoxy]cyclosporin.-   N 3-[2-(aminopropoxy)]cyclosporin.    The Compound Letters A to N are used hereafter.

Salts of Compounds of formula (I) are novel and as such form a furtherfeature of the present invention. In one embodiment the salt is selectedfrom the group consisting of hydrochloride, sulfate, phosphate,carbonate, acetate, tartrate, citrate, maleate, succinate, lactate,stearate, propionate, benzoate, fumarate, hippurate, gluconate,ascorbate, adipate, glutamate, mesylate, tosylate, oleate,laurylsulphate, hydrobromide and nitrate.

In certain embodiments, the cyclosporin derivatives provided herein havean optimized potency for immune suppression. The immunomodulationpotency relative to cyclosporin A can be measured in an assay of themixed lymphocyte response (MLR). The assay is known to one of skill inthe art, for example, see, U.S. Pat. No. 6,946,465. Lymphocytes fromrats, mouse or human could be used in the assay. In certain embodiments,the cyclosporin derivatives provided herein are equipotent tocyclosporin A in the MLR assay. In certain embodiments, the cyclosporinderivatives provided herein are from about 1 to about 5000, from about10 to about 3000, from about 50 to about 2000, from about 70 to about1000, from about 100 to about 500, from about 150 to about 300 fold lesspotent than cyclosporin A. In certain embodiments, the cyclosporinderivatives provided herein are from about 10 to about 100 or about 100to about 5000 fold less potent than cyclosporin A. In certainembodiments, the cyclosporin derivatives provided herein are from about2 to about 100, from about 2 to about 80, from about 2 to about 65, fromabout 2 to about 50, from about 2 to about 30, from about 2 to about 20,from about 2 to about 10, from about 2 to about 9, from about 2 to about7 or from about 2 to about 5 fold less potent than cyclosporin A. Incertain embodiments, the cyclosporin derivatives provided herein areabout 1, 5, 10, 15, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500or 1000 fold less potent than cyclosporin A.

Other cell-based assays can also be used to determine the activityagainst production of specific pro-inflammatory cytokines, including,IL-1, IL-2, IL-4, IL-6, TNF-α, interferons and TGF-β. In certainembodiments, the cyclosporin derivatives herein provide clinical benefitin the treatment of the dry eye or uveitis by down regulating cytokines.

In certain embodiments, the cyclosporin derivatives provided herein havesolubility that is useful for creating an optimized formulation fordelivery and release of the compound in eye. In certain embodiments, thecyclosporin derivatives provided herein have improved solubility overcyclosporin A. In certain embodiments, the cyclosporin derivatives haveaqueous solubility from about 0.001 to about 5 mM, about 0.005 to about3 mM, about 0.007 to about 1 mM, about 0.01 to about 0.5 mM, about 0.02to about 0.3 mM, about 0.05 to about 0.1 mM, about 0.06 to about 0.6 mM,about 0.08 to about 0.5 mM or about 0.1 to about 1 mM.

In certain embodiments, the cyclosporin derivatives provided herein havean improved safety profile relative to cyclosporin. The safety profilecan be determined in a variety of test models including acute andchronic animal toxicology models. Potential human toxicity could also bedetermined using human-cell-based assays known as cytotoxicity assays.Cytotoxicity can be evaluated in various cell types using a variety ofindicators and reporters, for example, see, Guidance Document on UsingIn Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity,(NIH Publication No. 01-4500). In certain embodiments, the compounds foruse in the methods provided herein are from about 2 to about 10, fromabout 3 to about 8, from about 2 to about 6, from about 2 to about 5,from about 2 to about 3 fold less active than cyclosporin in acytotoxicity assay.

In certain embodiments, the compound is in a pure form. Purity can beany purity known to those of skill in the art such as absolute purity,stereochemical purity or both. In certain embodiments, the compound isat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%pure. In certain embodiments, the compound is at least 90% pure. Infurther embodiments, the compound is at least 98% pure. Methods ofpurifying these compounds are described below.

Compounds of Formula (I) or pharmaceutically acceptable salts thereofare known in the literature or may be prepared by the adaptation ofknown methods, for example as described in U.S. Pat. No. 6,583,265 orInternational Patent publication Nos. WO99/65933, WO99/67280, thecontents of which are incorporated herein by reference.

4.2.2 Pharmaceutical Compositions and Methods of Administration

The cyclosporin compounds for use in the methods provided herein areuseful for treatment of ocular diseases.

The methods provided herein use pharmaceutical compositions containingat least one compound of general Formula (I), if appropriate in the saltform, either used alone or in the form of a combination with one or morecompatible and pharmaceutically acceptable carriers, such as diluents oradjuvants, or in combination with other active ingredients. In clinicalpractice the cyclosporin compounds for use in the methods providedherein may be administered by any conventional route, including but notlimited to topically orally, parenterally or by inhalation (e.g. in theform of aerosols). In one embodiment, the cyclosporin compounds for usein the methods provided herein are administered orally.

Use may be made, as solid compositions for oral administration, oftablets, pills, hard gelatin capsules, powders or granules. In thesecompositions, the active product provided herein is mixed with one ormore inert diluents or adjuvants, such as sucrose, lactose or starch.

These compositions can comprise substances other than diluents, forexample a lubricant, such as magnesium stearate, or a coating intendedfor controlled release.

Use may be made, as liquid compositions for oral administration, ofsolutions which are pharmaceutically acceptable, suspensions, emulsions,syrups and elixirs containing inert diluents, such as water or liquidparaffin. These compositions can also comprise substances other thandiluents, for example wetting, sweetening or flavouring products.

The compositions for parenteral administration can be emulsions orsterile solutions. Use may be made, as solvent or vehicle, of propyleneglycol, a polyethylene glycol, vegetable oils, in particular olive oil,or injectable organic esters, for example ethyl oleate. Thesecompositions can also contain adjuvants, in particular wetting,isotonizing, emulsifying, dispersing and stabilizing agents.Sterilization can be carried out in several ways, for example using abacteriological filter, by radiation or by heating. They can also beprepared in the form of sterile solid compositions which can bedissolved at the time of use in sterile water or any other injectablesterile medium.

The compositions can also be aerosols. For use in the form of liquidaerosols, the compositions can be stable sterile solutions or solidcompositions dissolved at the time of use in apyrogenic sterile water,in saline or any other pharmaceutically acceptable vehicle. For use inthe form of dry aerosols intended to be directly inhaled, the activeprinciple is finely divided and combined with a water-soluble soliddiluent or vehicle, for example dextran, mannitol or lactose.

In certain embodiments, a composition provided herein is apharmaceutical composition or a single unit dosage form. Pharmaceuticalcompositions and single unit dosage forms provided herein comprise aprophylactically or therapeutically effective amount of one or moreprophylactic or therapeutic agents (e.g., a compound provided herein, orother prophylactic or therapeutic agent), and a typically one or morepharmaceutically acceptable carriers or excipients. In a specificembodiment and in this context, the term “pharmaceutically acceptable”means approved by a regulatory agency of the Federal or a stategovernment or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund'sadjuvant (complete and incomplete)), excipient, or vehicle with whichthe therapeutic is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. In certain embodiments, water is acarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents.

Lactose free compositions provided herein can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose freecompositions comprise an active ingredient, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Exemplary lactose free dosage forms comprise an activeingredient, microcrystalline cellulose, pre gelatinized starch, andmagnesium stearate.

Further provided are anhydrous pharmaceutical compositions and dosageforms comprising active ingredients, since water can facilitate thedegradation of some compounds. For example, the addition of water (e.g.,5%) is widely accepted in the pharmaceutical arts as a means ofsimulating long term storage in order to determine characteristics suchas shelf life or the stability of formulations over time. See, e.g.,Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed.,Marcel Dekker, NY, N.Y., 1995, pp. 379 80. In effect, water and heataccelerate the decomposition of some compounds. Thus, the effect ofwater on a formulation can be of great significance since moistureand/or humidity are commonly encountered during manufacture, handling,packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms provided hereincan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are, in certainembodiments, anhydrous if substantial contact with moisture and/orhumidity during manufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are, in certain embodiments, packaged using materials knownto prevent exposure to water such that they can be included in suitableformulary kits. Examples of suitable packaging include, but are notlimited to, hermetically sealed foils, plastics, unit dose containers(e.g., vials), blister packs, and strip packs.

Further provided herein are pharmaceutical compositions and dosage formsthat comprise one or more compounds that reduce the rate by which anactive ingredient will decompose. Such compounds, which are referred toherein as “stabilizers,” include, but are not limited to, antioxidantssuch as ascorbic acid, pH buffers, or salt buffers.

The pharmaceutical compositions and single unit dosage forms can takethe form of solutions, suspensions, emulsion, tablets, pills, capsules,powders, sustained-release formulations and the like. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Such compositions and dosage forms willcontain a prophylactically or therapeutically effective amount of aprophylactic or therapeutic agent, in certain embodiments, in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject. The formulation shouldsuit the mode of administration. In one embodiment, the pharmaceuticalcompositions or single unit dosage forms are sterile and in suitableform for administration to a subject, such as an animal subject, or amammalian subject, and such as a human subject.

A pharmaceutical composition provided herein is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, intramuscular, subcutaneous,oral, buccal, sublingual, inhalation, intranasal, transdermal, topical,transmucosal, intra-tumoral and intra-synovial. In a specificembodiment, the composition is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenous,subcutaneous, intramuscular, oral, intranasal or topical administrationto human beings. In an embodiment, a pharmaceutical composition isformulated in accordance with routine procedures for subcutaneousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocamne to ease pain at the site of theinjection.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a subject,including suspensions (e.g., aqueous or non aqueous liquid suspensions,oil in water emulsions, or a water in oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a subject; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a subject.

The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. For example, a dosage form usedin the initial treatment of ocular disease may contain larger amounts ofone or more of the active ingredients it comprises than a dosage formused in the maintenance treatment of the disease. Similarly, aparenteral dosage form may contain smaller amounts of one or more of theactive ingredients it comprises than an oral dosage form used to treatthe same disease or disorder. These and other ways in which specificdosage forms encompassed herein will vary from one another will bereadily apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Generally, the ingredients of compositions provided herein are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Typical dosage forms provided herein comprise a compound providedherein, or a pharmaceutically acceptable salt, solvate or hydratethereof lie within the range of from about 0.1 mg to about 1000 mg perday, given as a single once-a-day dose in the morning or as divideddoses throughout the day taken with food. Particular dosage formsprovided herein have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0,10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of theactive cyclosporin.

4.2.2.1 Transdermal, Topical & Mucosal Dosage Forms

In certain embodiments, provided herein are transdermal, topical, andmucosal dosage forms. Transdermal, topical, and mucosal dosage formsprovided herein include, but are not limited to, ophthalmic solutions,sprays, aerosols, creams, lotions, ointments, gels, solutions,emulsions, suspensions, or other forms known to one of skill in the art.See, e.g., Remington's Pharmaceutical Sciences, 20th ed., MackPublishing, Easton Pa. (2000); and Introduction to Pharmaceutical DosageForms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage formssuitable for treating mucosal tissues within the oral cavity can beformulated as mouthwashes or as oral gels. Further, transdermal dosageforms include “reservoir type” or “matrix type” patches, which can beapplied to the skin and worn for a specific period of time to permit thepenetration of a desired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed herein are well known to those skilled in thepharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3diol, isopropyl myristate, isopropyl palmitate, mineral oil, andmixtures thereof to form lotions, tinctures, creams, emulsions, gels orointments, which are non toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients provided herein. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide;dimethylacetamide; dimethylformamide; polyethylene glycol; pyrrolidonessuch as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone);urea; and various water soluble or insoluble sugar esters such as Tween80 (polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery enhancing orpenetration enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

4.2.2.2 Pharmaceutical Compositions for Ophthalmic Administration

The methods provided herein use pharmaceutical compositions containingat least one compound of general Formula (I), if appropriate in the saltform, either used alone or in the form of a combination with one or morecompatible and pharmaceutically acceptable carriers, such as diluents oradjuvants, or in combination with other active ingredient, such asanother agent useful in treatment of ocular diseases. In clinicalpractice the pharmaceutical compositions containing cyclosporincompounds for use in the methods provided herein may be administered inany form suitable for ocular drug administration, e.g., as a solution,suspension, ointment, gel, liposomal dispersion, colloidal microparticlesuspension, injection, such as intraocular injection or subconjuctivalinjection or the like, or in an ocular insert, e.g., in an optionallybiodegradable controlled release polymeric matrix. In certainembodiments, the compositions are formulated as aqueous solutions. Inother embodiments, the compositions are suspensions, viscous orsemi-viscous gels, or other types of solid or semi-solid compositions.

A variety of carriers may be used in the formulations, including, butnot limited to water, mixtures of water and water-miscible solvents,such as C₁-C₇-alkanols, vegetable oils or mineral oils comprising from0.5 to 5% non-toxic water-soluble polymers, natural products, such asgelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum,carrageenin, agar and acacia, starch derivatives, such as starch acetateand hydroxypropyl starch, and also other synthetic products, such aspolyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether,polyethylene oxide, cross-linked polyacrylic acid, such as neutralCarbopol, or mixtures of those polymers. The concentration of thecarrier is, typically, from 1 to 10000 times the concentration of theactive ingredient.

Additional ingredients that may be included in the formulation includetonicity enhancers, preservatives, solubilizers, non-toxic excipients,demulcents, sequestering agents, pH adjusting agents, co-solvents andviscosity building agents.

In certain embodiments, for the adjustment of the pH, for example, to aphysiological pH, buffers are used. The pH is typically maintainedwithin the range of about 4.0 to 8.0, about 4.0 to 6.0 or about 6.5 to7.5. In certain embodiments, the pH is maintained at about 6, 6.3, 6.5,6.7, 7.0, 7.2 or 7.5. Suitable buffers may be added, such as acetate,ascorbate, boric acid, sodium borate, potassium citrate, citric acid,sodium bicarbonate, gluconate, lactate, phosphate, propionate, TRIS, andvarious mixed phosphate buffers (including combinations of Na₂HPO₄,NaH₂PO₄ and KH₂PO₄) and mixtures thereof. The amount of buffer substanceadded is, for example, that necessary to ensure and maintain aphysiologically tolerable pH range. Generally, buffers will be used inamounts ranging from about 0.05 to 3% by weight, about 0.07 to 2.5% byweight, about 0.1 to 2% by weight, about 0.5 to 2% by weight or about 1to 2% by weight. In certain embodiments, the amount of buffer is about0.05, 0.1, 0.5, 1, 1.5, 2, 2.3 or 3% by weight of the formulation.

In certain embodiments, the compositions contain tonicity enhancingagents selected from ionic and non-ionic compounds. Ionic compounds foruse herein include, but are not limited to alkali metal or alkalineearth metal halides, such as, for example, CaCl₂, KBr, KCl, LiCl, Nal,NaBr or NaCl, or boric acid. Non-ionic tonicity enhancing agents are,for example, urea, glycerol, sorbitol, mannitol, propylene glycol, ordextrose. The compositions provided herein contain, for example,sufficient tonicity enhancing agent to impart to the ready-for-useophthalmic composition an osmolality of approximately from about 50 to1000 mOsmol, from about 100 to 500 mOsmol, from about 200 to 400 mOsmol,from about 280 to 350 mOsmol or from about 230 to 320 mOsmol.

In certain embodiments, the compositions provided herein contain apreservative. Examples of preservatives are quaternary ammonium salts,such as cetrimide, benzalkonium chloride or benzoxonium chloride,alkyl-mercury salts of thiosalicylic acid, such as, for example,thiomersal, phenylmercuric nitrate, phenylmercuric acetate orphenylmercuric borate, parabens, such as, for example, methylparaben orpropylparaben, alcohols, such as, for example, chlorobutanol, benzylalcohol or phenyl ethanol, guanidine derivatives, such as, for example,chlorohexidine or polyhexamethylene biguanide, sodium perborate,Germa®II or sorbic acid. In certain embodiments, the preservative iscetrimide, benzalkonium chloride, benzoxonium chloride or parabens.Where appropriate, a sufficient amount of preservative is added to theophthalmic composition to ensure protection against secondarycontaminations during use caused by bacteria and fungi.

In another embodiment, the topical formulations provided herein do notinclude a preservative. Such formulations would be useful for patientswho wear contact lenses, or those who use several topical ophthalmicdrops and/or those with an already compromised ocular surface whereinlimiting exposure to a preservative may be more desirable.

The topical formulation provided herein can additionally contain asolubilizer. A solubilizer suitable for use in the composition is forexample selected from tyloxapol, fatty acid glycerol polyethylene glycolesters, fatty acid polyethylene glycol esters, polyethylene glycols,glycerol ethers, a cyclodextrin (for example alpha-, beta- orgamma-cyclodextrin, e.g. alkylated, hydroxyalkylated, carboxyalkylatedor alkyloxycarbonyl-alkylated derivatives, or mono- ordiglycosyl-alpha-, beta- or gamma-cyclodextrin, mono- ordimaltosyl-alpha-, beta- or gamma-cyclodextrin or panosyl-cyclodextrin),polysorbate 20, polysorbate 80, olive oil, castor oil, arachis oil,mineral oil or mixtures of those compounds. In certain embodiments, thesolubilizer is a reaction product of castor oil and ethylene oxide, forexample the commercial products Cremophor® EL or Cremophor RH40®.Reaction products of castor oil and ethylene oxide are known to beparticularly good solubilizers that are tolerated well by the eye. Inother embodiments, the solubilizer is selected from tyloxapol and from acyclodextrin. The concentration of the solubilized used herein dependsespecially on the concentration of the active ingredient. The amountadded is typically sufficient to solubilize the active ingredient. Forexample, the concentration of the solubilizer is from 0.1 to 5000 timesthe concentration of the active ingredient.

The formulations can further contain non-toxic excipients, such as, forexample, petroleum jelly, dimethyl sulfoxide, Miglyol 182 (commerciallyavailable from Dynamit Nobel Kay-Fries Chemical Company, Mont Vale,N.J.), an alcohol (e.g. ethanol, n-propyl alcohol, or isopropylalcohol), liposomes or liposome-like products or a silicone fluid,emulsifiers, wetting agents or fillers, such as, for example, thepolyethylene glycols designated 200, 300, 400 and 600, or Carbowaxdesignated 1000, 1500, 4000, 6000 and 10000. Other excipients that maybe used if desired are listed below but they are not intended to limitin any way the scope of the possible excipients. They are complexingagents, such as disodium-EDTA or EDTA, antioxidants, such as ascorbicacid, acetylcysteine, cysteine, sodium hydrogen sulfite,butyl-hydroxyanisole, butyl-hydroxytoluene or α-tocopherol acetate;stabilizers, such as a cyclodextrin, thiourea, thiosorbitol, sodiumdioctyl sulfosuccinate or monothioglycerol, vitamin E and vitamin Ederivatives, such as Vitamin E Tocopherol Polyethylene Glycol 1000Succinate (TPGS); or other excipients, such as, for example, lauric acidsorbitol ester, triethanol amine oleate or palmitic acid ester. Incertain embodiments, the excipients are complexing agents, such asdisodium-EDTA and stabilizers, such as a cyclodextrin. The amount andtype of excipient added is in accordance with the particularrequirements and is generally in the range of from approximately 0.0001to approximately 90% by weight.

Other compounds may also be added to the formulations provided herein toincrease the viscosity of the carrier. Examples of viscosity enhancingagents include, but are not limited to: cellulosic polymers such asmethylcellulose (MC), hydroxyethylcellulose (HEC),hydroxypropylcellulose (HPC), hydroxypropyl-methylcellulose (HPMC), andsodium carboxymethylcellulose (NaCMC), and other swellable hydrophilicpolymers such as polyvinyl alcohol (PVA), hyaluronic acid or a saltthereof (e.g., sodium hyaluronate), chondroitin sulfate and its salts,dextrans and crosslinked acrylic acid polymers commonly referred to as“carbomers” (and available from B.F. Goodrich as Carbopol® polymers). Incertain embodiments, amount of any thickener is such that a viscosity inthe range of about 15 cps to 25 cps is provided, as a solution having aviscosity in the aforementioned range is generally considered optimalfor both comfort and retention of the formulation in the eye.

In certain embodiments, the compositions provided herein comprise acompound provided herein, carmellose sodium in purified water as alubricant (purified water as a lubricant), hypotonic, borate buffer,sodium chloride, potassium chloride, sodium bicarbonate, calciumchloride, magnesium chloride and sodium phosphate. In certainembodiments, the compositions provided herein comprise a compoundprovided herein mineral oil and lanolin. In certain embodiments, thecompositions provided herein comprise a compound provided herein,buffered solution, isotonic aqueous solution, citrate buffer, sodiumchloride, edetate disodium and polyquad polyquaternium. In certainembodiments, the compositions provided herein comprise a compoundprovided herein, sodium chloride, boric acid, potassium chloride,calcium chloride, magnesium chloride, purified water, and stabilizedoxychloro complex.

4.2.3 Methods of Treatment

Provided herein are methods for treating or preventing ocular disease,such as dry eye, uveitis and phacoanaphylactic endophthalmitis in asubject in need of such treatment.

Although it appears that dry eye may result from a number of unrelatedpathogenic causes, all presentations of the complication share a commoneffect, which is the breakdown of the pre-ocular tear film, whichresults in dehydration of the exposed outer surface and many of thesymptoms outlined above. In certain embodiments, symptoms of dry eyeinclude feeling of dryness, and persistent irritation such as is oftencaused by small bodies lodging between the eye lid and the eye surface.In severe cases, vision may be substantially impaired. Dry eye diseaseincludes, but is not limited to keratoconjunctivitis sicca (KCS),age-related dry eye, Stevens-Johnson syndrome, Sjogren's syndrome,ocular cicatrical pemphigoid, blepharitis, corneal injury, infection,Riley-Day syndrome, congenital alacrima, nutritional disorders ordeficiencies (including vitamin), pharmacologic side effects, eye stressand glandular and tissue destruction, environmental exposure to smog,smoke, excessively dry air, airborne particulates, autoimmune and otherimmunodeficient disorders. The methods provided herein treat or preventone or more diseases of dry eye.

In certain embodiments, provided herein are methods for treatment orprevention of uveitis. In certain embodiments, uveitis is anterior,intermediate, posterior, or diffuse uveitis. Anterior uveitis islocalized primarily to the anterior segment of the eye and includesiritis and iridocyclitis. Intermediate uveitis, also called peripheraluveitis, is centered in the area immediately behind the iris and lens inthe region of the ciliary body and pars plana. Posterior uveitissignifies any of a number of forms of retinitis, choroiditis, or opticneuritis. Diffuse uveitis implies inflammation involving all parts ofthe eye, including anterior, intermediate, and posterior structures. Thesymptoms of uveitis can vary depending on the location of the uveitis;acute and severe symptoms are generally more common in anterior uveitisand can include: eye pain, eye redness, photophobia, blurred ordecreased vision and blindness. Other symptoms include “floaters” whichare small specks or clouds that move with the field of vision, chronicflare in the eye, band keratopathy, secondary glaucoma and posteriorsubcapsular cataracts.

In certain embodiments, provided herein are methods for treatment orprevention of phacoanaphylactic endophthalmitis. In certain embodiments,provided herein are methods for treatment or prevention ofphacoanaphylaxis, which is a severe form of uveitis.

4.2.4 Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the dosage regimenwhich he considers most appropriate according to a preventive orcurative treatment and according to the age, weight, stage of thedisease and other factors specific to the subject to be treated. Incertain embodiments, dose rates are from about 0.001 to about 1000 mgper day for an adult. In certain embodiments, doses are from about 1 toabout 1000 mg per day for an adult, or from about 5 to about 250 mg perday or from about 10 to 50 mg per day for an adult. In certainembodiments, doses are from about 5 to about 400 mg per day, about 25 to200 mg per day, about 50 to about 500 mg per day per adult. In certainembodiments, doses are about 0.001 to about 500 mg per day, about 0.001to about 100 mg per day, from about 0.005 to about 50 mg per day, fromabout 0.01 to 10 mg per day, from about 0.03 to 1 mg per day, from about0.05 to 1 mg per day, from about 0.06 to 1 mg per day, from about 0.08to 1 mg or from about 0.1 to 1 mg per day for an adult. In certainembodiments, doses are about 0.001 mg, 0.005 mg, 0.01 mg, 0.02 mg, 0.03mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.3mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg,45 mg, 50 mg, 60 mg, 70 mg, 80 mg or 100 mg per day per adult.

The amount of the compound or composition provided herein which will beeffective in the prevention or treatment of a disorder or one or moresymptoms thereof will vary with the nature and severity of the diseaseor condition, and the route by which the active ingredient isadministered. The frequency and dosage will also vary according tofactors specific for each subject depending on the specific therapy(e.g., therapeutic or prophylactic agents) administered, the severity ofthe disorder, disease, or condition, the route of administration, aswell as age, body, weight, response, and the past medical history of thesubject. Effective doses may be extrapolated from dose-response curvesderived from in vitro or animal model test systems.

Exemplary doses of a composition include milligram or microgram amountsof the active compound per kilogram of subject or sample weight (e.g.,from about 0.1 micrograms per kilogram to about 50 milligrams perkilogram, from about 0.5 micrograms per kilogram to about 25 milligramsper kilogram, from about 0.1 micrograms per kilogram to about 10milligrams per kilogram, from about 1 micrograms per kilogram to about 5milligrams per kilogram, from about 10 micrograms per kilogram to about5 milligrams per kilogram, from about 100 micrograms per kilogram toabout 2.5 milligrams per kilogram, or from about 100 microgram perkilogram to about 1 milligrams per kilogram). In certain embodiments,the doses are about 10 micrograms per kilogram to about 50 milligramsper kilogram, about 100 micrograms per kilogram to about 25 milligramsper kilogram, or about 100 microgram per kilogram to about 10 milligramsper kilogram of subject or sample weight. For compositions provided, thedosage administered to a subject is typically 0.140 mg/kg to 3 mg/kg ofthe subject's body weight, based on weight of the active compound. Insome embodiments, the dosage administered to a subject is between 0.20mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50 mg/kg of thesubject's body weight. In certain embodiments, for compositions providedherein, the dosage administered to a subject is from about 0.0001 mg/kgto 0.01 mg/kg of the subject's body weight, based on weight of theactive compound. In certain embodiments, for compositions providedherein, the dosage administered to a subject is from about 0.0005 mg/kgto 0.01 mg/kg of the subject's body weight, based on weight of theactive compound. In certain embodiments, for compositions providedherein, the dosage administered to a subject is 0.140 mg/kg to 3 mg/kgof the subject's body weight, based on weight of the active compound. Incertain embodiments, the dosage administered to a subject is from about0.0001 mg/kg to about 0.01 mg/kg, about 0.0005 mg/kg to about 0.001mg/kg, from about 0.001 mg/kg to about 0.01 mg/kg, about 0.20 mg/kg and2.00 mg/kg, or between 0.30 mg/kg and 1.50 mg/kg of the subject's bodyweight. In certain embodiments, the dosage administered to a subject isabout 0.001 mg/kg, such that for a patient weighing about 60 kg, theamount of the compound of formula I administered is about 0.006 mg perdose.

In certain embodiments, the recommended daily dose range of acomposition provided herein for the conditions described herein liewithin the range of from about 0.0005 mg to about 0.1 mg per day or fromabout 0.1 mg to about 1000 mg per day, given as a single once-a-day doseor as divided doses throughout a day. In one embodiment, the daily doseis administered twice daily in equally divided doses. In one embodiment,a daily dose range is from about 10 mg to about 200 mg per day, about 10mg and about 150 mg per day, or about 25 and about 100 mg per day. Inanother embodiment, a daily dose range is from about 0.0001 mg to about0.01 mg per day, in another embodiment, a daily dose range is from about0.0005 mg to about 0.005 mg per day, or in another embodiment, a dailydose range is from about 0.001 mg to about 0.05 mg per day. It may benecessary to use dosages of the active ingredient outside the rangesdisclosed herein in some cases, as will be apparent to those of ordinaryskill in the art. Furthermore, it is noted that the clinician ortreating physician will know how and when to interrupt, adjust, orterminate therapy in conjunction with subject response.

Different therapeutically effective amounts may be applicable fordifferent diseases and conditions, as will be readily known by those ofordinary skill in the art. Similarly, amounts sufficient to prevent,manage, treat or ameliorate such disorders, but insufficient to cause,or sufficient to reduce, adverse effects associated with the compositionprovided herein are also encompassed by the above described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In a specific embodiment, the dosage of the composition provided herein,based on weight of the active compound, administered to prevent, treat,manage, or ameliorate a disorder, or one or more symptoms thereof in asubject is about 0.0001 mg/kg, 0.0005 mg/kg, 0.0007 mg/kg, 0.0009 mg/kg,0.001 mg/kg, 0.0012 mg/kg, 0.0015 mg/kg, 0.0017 mg/kg, 0.002 mg/kg,0.0025 mg/kg, 0.003 mg/kg, 0.0035 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.01mg/kg, 0.1 mg/kg, 1 mg/kg or more of a subject's body weight. In anotherembodiment, the dosage of the composition provided herein isadministered to prevent, treat, manage, or ameliorate a disorder, or oneor more symptoms thereof in a subject is a unit dose of from about0.0001 mg to about 100 mg, 0.0005 mg to 50 mg, 0.0008 mg to 25 mg, 0.001mg to 10 mg, 0.0013 mg to 10 mg, 0.0015 mg to 10 mg, 0.0018 mg to 10 mg,0.002 mg to 10 mg, 0.005 mg to 10 mg, 0.015 mg to 10 mg, 0.01 mg to 1 mgor 0.1 mg to 1 mg.

In certain embodiments, administration of the same composition providedherein may be repeated and the administrations may be separated by atleast 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days,2 months, 75 days, 3 months, or 6 months. In other embodiments,administration of the same prophylactic or therapeutic agent may berepeated and the administration may be separated by at least at least 1day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2months, 75 days, 3 months, or 6 months.

In certain aspects, provided are unit dosages comprising a compoundprovided herein, or a pharmaceutically acceptable salt thereof, in aform suitable for administration. Such forms are described in detail.above. In particular embodiments, the unit dosages comprise about0.001, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.015, 0.02, 0.025,0.03, 0.05, 0.1, 1, 5, 10, 25, 50 or 100 mg active ingredient. Such unitdosages can be prepared according to techniques familiar to those ofskill in the art.

4.2.5 Combination Therapy

In certain embodiments, provided herein are methods of treatment ofprevention that comprise the administration of a second agent effectivefor the treatment or prevention of ocular diseases and disorders such asaqueous deficient dry-eye state, uveitis or phacoanaphylacticendophthalmitis in a subject in need thereof. The second agent can beany agent known to those of skill in the art to be effective for thetreatment or prevention of dry-eye, uveitis or phacoanaphylacticendophthalmitis. The second agent can be a second agent presently knownto those of skill in the art, or the second agent can be second agentlater developed for the treatment or prevention of dry-eye, uveitisand/or phacoanaphylactic endophthalmitis. In certain embodiments, thesecond agent is presently approved for the treatment or prevention ofdry eye, uveitis and/or phacoanaphylactic endophthalmitis.

In certain embodiments, the second therapeutically active agent could beany drug which might be useful in treating the symptoms of dry eye,uveitis and/or phacoanaphylactic endophthalmitis, or any of itsunderlying causes. In addition, the second therapeutically active agentcould be any drug which is useful in preventing or treating any diseasewhich might occur simultaneously to dry eye, uveitis and/orphacoanaphylactic endophthalmitis disease, whether or not the disease isrelated. In certain embodiments, the second therapeutically active agentcould be a drug which is used in topical ophthalmic compositions whichmight cause, contribute to, or aggravate ocular diseases contemplatedherein as a side effect of its use. In this aspect, the methods providedherein are useful in reducing or eliminating said side effect.

One class of useful second therapeutically active agents in the methodsfor treating ocular disease provided herein is nucleotide purinergicreceptor agonists such as uridine 5′-triphosphate, dinucleotides,cytidine 5′-diphosphosphate, adenosine 5′-diphosphate, P¹-(cytidine5′-)-P-(uridine 5′-)tetraphosphates, P¹, P⁴-di(uridine5′)-tetraphosphates, or their therapeutically effective analogues orderivatives, which may affect tear secretion, particularly the mucouslayer of tears, and thus may have potential in treating dry eye disease.These compounds are described in the following patents, all of which areincorporated herein by reference: U.S. Pat. No. 6,555,675; U.S. Pat. No.6,548,658; U.S. Pat. No. 6,436,910; U.S. Pat. No. 6,348,589; U.S. Pat.No. 6,331,529; U.S. Pat. No. 6,323,187; U.S. Pat. No. 6,319,908; andU.S. Pat. No. 5,900,407.

Another useful class of compounds that are useful as secondtherapeutically active agents in the methods for treating ocular diseaseprovided herein is nicotinic receptor agonists such as nicotine and itsanalogs, trans-metanicotine and its analogs, epibatidine and itsanalogs, pyridol derivatives, piperidine alkaloids such as lobeline andits analogs, certain para-alkylthiophenol derivatives, and imidaclopridand its analogs. These compounds are believed to stimulate secretion ofmucin by the conjunctival goblet cells, and thus may be useful intreating dry eye, as disclosed in U.S. Pat. No. 6,277,855, which isincorporated herein by reference.

Another useful class of second therapeutically active agents in themethods for treating ocular disease provided herein is tetracycline,derivatives or analogues of tetracycline, or chemically modifiedtetracycline. These compounds are believed to have potential incorrecting delayed tear clearance, as described in U.S. Pat. No.6,455,583 B1, incorporated herein by reference, which is related to somecases of dry eye.

Another class of compounds that are useful as second therapeuticallyactive agents in the methods for treating ocular disease provided hereinis corticosteroids such as methylprednisolone sodium succinate,prednisolone acetate, prednisolone sodium phosphate, fluorometholone,fluorometholone acetate, dexamethasone sodium phosphate,hydroxymethylprogesterone, rimexolane, budesonide, and tixocortolpivalatein, which are believed to be useful in treating dry eye asdisclosed in U.S. Pat. No. 6,153,607, incorporated herein by reference.

Another class of compounds which are useful as second therapeuticallyactive agents in the methods for treating ocular disease provided hereinis products of human lacrimal gland acinar epithelia such as growthfactors or cytokines including the transforming growth factor beta(TGFβ), which are disclosed to be useful in treating dry eye in U.S.Pat. No. 5,652,209, incorporated herein by reference.

Another class useful second therapeutically active agents in the methodsfor treating ocular disease provided herein is androgens or androgenanalogues such as 17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one,testosterone or testosterone derivatives, 4,5α-dihydrotestosterone orderivatives, 17β-hydroxy-5α-androstane and derivatives,19-nortestosterone or derivatives, and nitrogen-substituted androgens,which are taught to be useful in treating dry eye disease in thefollowing patents which are incorporated herein by reference, U.S. Pat.No. 6,107,289; U.S. Pat. No. 5,958,912; U.S. Pat. No. 5,688,765; andU.S. Pat. No. 5,620,921.

In certain embodiments, the second therapeutically active agents in themethods for treating ocular disease provided herein are topicalcorticosteroids, including, but not limited to prednisone andtriamcinolone acetonide; cycloplegic agents such as cyclopentolate andhomatropine hydrobromide and immunosuppressive drugs, such asmethotrexate, cyclosporin A, cyclophosphamide and chlorambucil.

Another class useful second therapeutically active agents in the methodsfor treating ocular disease provided herein are antibiotics, including,but not limited to macrolides (e.g. rapamycin, tobramycin, ascomycin,azalides such as azithromycin); oxazolidinones (e.g. linezolid,eperezolid); quinolones (e.g. ofloxacin, norfloxacin, ciprofloxacin,lomefloxacin), gentamicin, and pilocarpine.

Other useful second therapeutically active agents in the methods fortreating ocular disease provided herein are selected from cyclosporin A,cyclosporin B, cyclosporin C, cyclosporin D, and cyclosporin G.

In certain embodiments, the second agent can be formulated or packagedwith the cyclosporin derivatives provided herein. Of course, the secondagent will only be formulated with the cyclosporin derivative providedherein when, according to the judgment of those of skill in the art,such co-formulation should not interfere with the activity of eitheragent or the method of administration. In certain embodiment, thecyclosporin derivative provided herein and the second agent areformulated separately. They can be packaged together, or packagedseparately, for the convenience of the practitioner of skill in the art.

The dosages of the second agents are to be used in the combinationtherapies provided herein. In certain embodiments, dosages lower thanthose which have been or are currently being used to prevent or treatthe diseases contemplated herein are used in the combination therapiesprovided herein. The recommended dosages of second agents can obtainedfrom the knowledge of those of skill. For those second agents that areapproved for clinical use, recommended dosages are described in, forexample, Hardman et al., eds., 1996, Goodman & Gilman's ThePharmacological Basis Of Basis Of Therapeutics 9^(th) Ed, Mc-Graw-Hill,New York; Physician's Desk Reference (PDR) 57^(th) Ed., 2003, MedicalEconomics Co., Inc., Montvale, N.J., which are incorporated herein byreference in its entirety.

In various embodiments, the therapies (e.g., the cyclosporin derivativeprovided herein and the second agent) are administered less than 5minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hourapart, at about 1 to about 2 hours apart, at about 2 hours to about 3hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In certainembodiments, two or more therapies are administered within the samepatent visit.

In certain embodiments, the cyclosporin derivative provided herein andthe second agent are cyclically administered. Cycling therapy involvesthe administration of a first therapy (e.g., a first prophylactic ortherapeutic agents) for a period of time, followed by the administrationof a second therapy (e.g., a second prophylactic or therapeutic agents)for a period of time, followed by the administration of a third therapy(e.g., a third prophylactic or therapeutic agents) for a period of timeand so forth, and repeating this sequential administration, i.e., thecycle in order to reduce the development of resistance to one of theagents, to avoid or reduce the side effects of one of the agents, and/orto improve the efficacy of the treatment.

In certain embodiments, administration of the same agent may be repeatedand the administrations may be separated by at least 1 day, 2 days, 3days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In other embodiments, administration of the sameagent may be repeated and the administration may be separated by atleast at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days,45 days, 2 months, 75 days, 3 months, or 6 months.

In certain embodiments, a cyclosporin derivative provided herein and asecond agent are administered to a patient, such as a mammal, includinga human, in a sequence and within a time interval such that thecyclosporin derivative can act together with the other agent to providean increased benefit than if they were administered otherwise. Forexample, the second active agent can be administered at the same time orsequentially in any order at different points in time; however, if notadministered at the same time, they should be administered sufficientlyclose in time so as to provide the desired therapeutic or prophylacticeffect. In one embodiment, the cyclosporin derivative and the secondactive agent exert their effects at times which overlap. Each secondactive agent can be administered separately, in any appropriate form andby any suitable route. In other embodiments, the cyclosporin derivativeis administered before, concurrently or after administration of thesecond active agent.

In various embodiments, the cyclosporin derivative and the second agentare administered less than about 1 hour apart, at about 1 hour apart, atabout 1 hour to about 2 hours apart, at about 2 hours to about 3 hoursapart, at about 3 hours to about 4 hours apart, at about 4 hours toabout 5 hours apart, at about 5 hours to about 6 hours apart, at about 6hours to about 7 hours apart, at about 7 hours to about 8 hours apart,at about 8 hours to about 9 hours apart, at about 9 hours to about 10hours apart, at about 10 hours to about 11 hours apart, at about 11hours to about 12 hours apart, no more than 24 hours apart or no morethan 48 hours apart. In other embodiments, the cyclosporin derivativeand the second agent are administered concurrently.

In other embodiments, the cyclosporin derivative and the second agentare administered at about 2 to 4 days apart, at about 4 to 6 days apart,at about 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart.

In certain embodiments, the cyclosporin derivative and the second agentare cyclically administered to a patient. Cycling therapy involves theadministration of a first agent for a period of time, followed by theadministration of a second agent and/or third agent for a period of timeand repeating this sequential administration. Cycling therapy can reducethe development of resistance to one or more of the therapies, avoid orreduce the side effects of one of the therapies, and/or improve theefficacy of the treatment.

In certain embodiments, the cyclosporin derivative and the second activeagent are administered in a cycle of less than about 3 weeks, about onceevery two weeks, about once every 10 days or about once every week. Onecycle can comprise the administration of a cyclosporin derivative andthe second agent by infusion over about 90 minutes every cycle, about 1hour every cycle, about 45 minutes every cycle. Each cycle can compriseat least 1 week of rest, at least 2 weeks of rest, at least 3 weeks ofrest. The number of cycles administered is from about 1 to about 12cycles, more typically from about 2 to about 10 cycles, and moretypically from about 2 to about 8 cycles.

In other embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecyclosporin derivative can work together with the second active agent.For example, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day.

The second agent can act additively or synergistically with thecyclosporin derivative. In one embodiment, a cyclosporin derivative isadministered concurrently with one or more second agents in the samepharmaceutical composition. In another embodiment, a cyclosporinderivative is administered concurrently with one or more second agentsin separate pharmaceutical compositions. In still another embodiment, acyclosporin derivative is administered prior to or subsequent toadministration of a second agent. Also contemplated is administration ofa cyclosporin derivative and a second agent by the same or differentroutes of administration, e.g., oral and parenteral. In certainembodiments, when a cyclosporin derivative is administered concurrentlywith a second agent that potentially produces adverse side effectsincluding, but not limited to, toxicity, the second active agent canadvantageously be administered at a dose that falls below the thresholdthat the adverse side effect is elicited.

4.2.6 Kits

In certain embodiments, provided are kits for use in methods oftreatment or prophylaxis of ocular diseases. The kits can include apharmaceutical compound or composition provided herein and instructionsproviding information to a health care provider regarding usage fortreating or preventing ocular disease. Instructions may be provided inprinted form or in the form of an electronic medium such as a floppydisc, CD, or DVD, or in the form of a website address where suchinstructions may be obtained. A unit dose of a compound or compositionprovided herein can include a dosage such that when administered to asubject, a therapeutically or prophylactically effective plasma level ofthe compound or composition can be maintained in the subject for atleast 1 day. In some embodiments, a compound or composition providedherein can be included as a sterile aqueous pharmaceutical compositionor dry powder (e.g., lyophilized) composition. In one embodiment, thecompound is according to Formula (I).

In some embodiments, suitable packaging is provided. As used herein,“packaging” refers to a solid matrix or material customarily used in asystem and capable of holding within fixed limits a compound orcomposition provided herein suitable for administration to a subject.Such materials include glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes and the like. If e-beam sterilizationtechniques are employed, the packaging should have sufficiently lowdensity to permit sterilization of the contents.

Kits provided herein may also comprise, in addition to the compound orcomposition provided herein, second agents or compositions comprisingsecond agents for use with compound or composition as described in themethods above.

The following Examples illustrate the preparation of compositions usedin the methods of the present invention. Specifically documented is the7 step synthesis of 3-[2-(pyrrolidin-1-yl)ethoxy]cyclosporin (compoundC) from cyclosporin A. Also shown is the synthesis of the phosphate saltof compound C. Compounds A, B, D, E, F, and G, along with theirphosphate salts, can be synthesized in an analogous manner usingdifferent amines during the penultimate reductive amination step.

Numerous modifications and variations of the claimed subject matter arepossible in view of the teachings herein and, therefore, are within thescope of the claimed subject matter.

5 EXAMPLES Example E1

To a solution of[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-(2-pyrrolidin-1-yl)ethoxy-Sar]³cyclosporinA (150 mg) in methanol (10 mL) was added 25 wt % sodium methoxide inmethanol (0.04 mL) and the resulting mixture was stirred at roomtemperature for 24 hours under nitrogen. Methanol was removed underreduced pressure and the residue was diluted with ethyl acetate, washedwith saturated ammonium chloride, brine, and dried over anhydrous sodiumsulfate. After solvent removal, the residue was purified usingpreparative liquid chromatography to yield 33 mg of3-[2-(pyrrolidin--yl)ethoxy]cyclosporin (Compound C). ¹H NMR peaks at5.89, 7.10, 7.15, 7.64 and 7.96 ppm. LCMS (ESI): calculated forC₆₈H₁₂₂N₁₂O₁₃: 1314, found 1315.2 (M+H)⁺.

By proceeding in a similar manner the following compounds were alsoprepared:

Compound ¹H NMR A 6.00, 7.16, 7.23, 7.71, 7.97; phosphate salt: 5.97,7.18 (2H), 7.70, 8.05 B 5.97, 7.15, 7.23, 7.69, 7.95; phosphate salt:5.94, 7.17 (2H), 7.68, 8.05 D 5.94, 7.09, 7.16, 7.66, 7.90; phosphatesalt: 5.99, 7.18 (2H), 7.69, 8.05 E 6.08, 7.15, 7.22, 7.73, 7.92 F 6.05,7.16, 7.21, 7.71, 7.96; phosphate salt: 5.98, 7.18 (2H), 7.70, 8.05 G6.04, 7.16, 7.21, 7.69, 7.99 H 5.91 (0.5H), 5.93 (0.5H), 7.10 (2H),7.63, 7.99; (mixture of phosphate salt: 5.90 (0.5H), 5.93 (0.5H),diastereomers) 7.12 (2H), 7.63, 8.05 I 6.03, 7.15, 7.21, 7.73, 7.94 J6.07, 7.15, 7.22, 7.73, 7.93 K 5.75, 6.99, 7.08, 7.67, 7.86 L 5.89,7.09, 7.15, 7.67, 7.91 M 5.90, 7.09, 7.15, 7.68, 7.91 N 5.93, 7.16,7.24, 7.72, 7.96

The phosphoric salt of Compound C was prepared as follows: Compound C(50 mg) was dissolved in a solvent mixture of acetonitrile (1 mL) andwater (1.0 mL) and to this solution was added an aqueous solution ofH₃PO₄ (0.1 M, 0.38 mL). The resulting mixture was mixed thoroughly andthen lyophilized for 28 h to yield 50 mg of the corresponding phosphoricsalt. ¹H NMR peaks at 5.92, 7.11, 7.63 and 8.01 ppm.

Reference Example 1

The aldehyde,[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-formylmethoxy-Sar]³cyclosporin A (290mg), was dissolved in methanol (15 mL) and to this solution were addedacetic acid (30 μL), pyrrolidine (50 μL), and sodium cyanoborohydride(30 mg). The resulting mixture was stirred at room temperatureovernight. The solvent was removed and the residue was purified usingsilica gel column chromatography to yield 154 mg of[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-(2-pyrrolidin-1-yl)ethoxy-Sar]³cyclosporinA. ¹H NMR peaks at 5.95, 7.08, 7.16, 7.60 and 7.90 ppm.

Reference Example 2

To a suspension of Dess-Martin periodinane (300 mg) in dichloromethane(30 mL) was added[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-hydroxymethylmethoxy-Sar]³cyclosporinA (600 mg) in dichloromethane (15 mL) and the resulting mixture wasstirred at room temperature for 1 h. The reaction mixture was dilutedwith MTBE (100 mL), washed with a 1:1 (volume/volume) mixture of 10%Na₂S₂O₃ and saturated NaHCO₃ (2×50 mL), saturated brine solution, anddried over anhydrous sodium sulphate. After solvent removal, 580 mg of[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-formylmethoxy-Sar]³cyclosporin A wasobtained.

Reference Example 3

Camphor sulfonic acid (1.0 g) was added to a solution of[3′-acetoxy-N-methyl-Bmt]¹[2′-acetoxy-Sar]³cyclosporin A (5.14 g) in asolvent mixture of THF (10 mL) and dry ethylene glycol (100 mL), and theresulting mixture was stirred at 50° C. for 5 hours. The reactionmixture was diluted with saturated NaHCO₃ (150 mL), water (200 mL), andextracted with ethyl acetate (3×150 mL). The combined organic extractswere washed with water (150 mL), saturated sodium chloride solution (150mL), and dried over anhydrous sodium sulphate. After solvent removal,2.66 g of[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-hydroxymethylmethoxy-Sar]³cyclosporinA was obtained. ¹H NMR peaks at 5.90, 7.26, 7.46, 8.03 and 8.55 ppm.

Reference Example 4

Mercury acetate (4.4 g) was added to a solution of[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-thiophenyl-Sar]³cyclosporin A (4.4 g)in glacial acetic acid (90 mL) and the resulting mixture was stirred for3 hours at 50° C. The solvent was then removed and the residue dissolvedin ethyl acetate (300 mL), washed with a saturated solution of sodiumhydrogen carbonate (150 mL) and then brine (150 mL), and dried overanhydrous sodium sulphate. After removal of the solvent, the crudeproduct was purified using silica gel column chromatography to yield5.14 g of [3′-acetoxy-N-methyl-Bmt]¹[2′-acetoxy-Sar]³cyclosporin A.

Reference Example 5

N,N-Dimethylaminopyridine (1.23 g), triethylamine (1.39 mL) and aceticanhydride (0.63 mL) were added to a solution of[(R)-2′-thiophenyl-Sar]³cyclosporin A (4.36 g) in dry dichloromethane(60 mL). The resulting mixture was stirred at room temperature for about2.5 days. The reaction mixture was then diluted with ethyl acetate,washed with water and brine and concentrated. The crude product waspurified by chromatography using a silica gel column, eluting with amixture of ethyl acetate and hexane to yield[3′-acetoxy-N-methyl-Bmt]¹[(R)-2′-thiophenyl-Sar]³cyclosporin A.

Reference Example 6

A solution of cyclosporin A (8.0 g) in dry t-butyl methyl ether (TBME,50 mL) was added to a suspension of sodium amide (7.0 g) in liquidammonia (200 mL) at −33° C. under inert atmosphere. The resultingmixture was stirred at −33° C. for 90 minutes under an inert atmosphere.Phenyl disulfide (25 g) was then added, and the reaction mixture wasstirred for an additional 2 hours at −33° C. under an inert atmosphere.The reaction was then quenched with solid ammonium chloride (17.5 g) andthe ammonia was evaporated. The reaction mixture was then diluted withTBME (250 ml) and water (250 mL), mixed thoroughly, and the layersseparated. The organic layer was washed with brine (250 mL) and thenconcentrated. The residue was purified by chromatography using a silicagel column eluting first with a mixture of ethyl acetate and heptane,and then with a mixture of methanol and ethyl acetate, to yield 4.36 gof [(R)-2′-thiophenyl-Sar]³cyclosporin A.

5.1 Example 1 Oral Dosage Forms—Formulation of an Oral Capsule

One or more of the compounds for use in the methods provided herein canbe formulated as a capsule. Such a capsule can comprise 10 to 100 mg ofthe compound and on or more excipients selected from the groupconsisting of microcrystalline cellulose, pregelatinized starch,lactose, sodium starch glycolate, crospovidone, povidone,hydroxypropylcellulose, magnesium stearate and silicon dioxide. Theresulting composition can be encapsulated with one or more standardencapsulation compositions such as gelatin or a plasticizer.

5.2 Example 2 Formulation of an Oral Liquid

One or more of the compounds for use in the methods provided herein canbe formulated as a salt in a syrup or elixir. The compound or compoundscan be at a total concentration of 5 to 50 mg/mL. The syrup or elixircan further comprise polyethylene glycol, propylene glycol, mixtures ofpolyethylene glycol, PEG 400, a block copolymer of ethylene oxide andpropylene oxide (e.g., poloxamer 407), polysorbate 20, ethanol, a sugar,citric acid and/or flavoring.

5.3 Example 3 Formulation of an Ophthalmic Emulsion

The compounds for use in the methods provided herein can be formulatedas ophthalmic emulsion. Such an emulsion can comprise in each mL, 0.05%of the compound provided herein and one or more excipients selected fromglycerin; castor oil; polysorbate 80; carbomer 1342; purified water andsodium hydroxide to adjust the pH. The resulting formulation can have anosmolality of 230 to 320 mOsmol/kg and a pH of 6.5-8.0.

5.4 Example 4 Formulation of Ophthalmic Drops

The compounds for use in the methods provided herein can be formulatedas ophthalmic drops. Such drops can comprise one or more compounds foruse in the methods provided herein and one or more excipients selectedfrom purified water as a lubricant, sterile solution hypotonic-boratebuffer, electrolytes found in natural tears, sodium chloride, potassiumchloride, sodium chloride, sodium bicarbonate, calcium chloride,magnesium chloride, sodium phoshpate, sodium perborate and phosphonicacid stabilizer.

The compounds for use in the methods provided herein can be formulatedas ophthalmic drops. Such drops can comprise one or more compounds foruse in the methods provided herein and one or more excipients selectedfrom purified water as a lubricant, sterile solution hypotonic-boratebuffer, electrolytes found in natural tears, sodium chloride, potassiumchloride, sodium chloride, sodium bicarbonate, calcium chloride,magnesium chloride, sodium phosphate, sodium perborate and phosphonicacid stabilizer.

The ophthalmic formulations of the present invention are convenientlypackaged in forms suitable for metered application, such as incontainers equipped with a dropper, to facilitate application to theeye. Containers suitable for dropwise application are usually made ofsuitable inert, non-toxic plastic material, and generally containbetween about 0.5 and about 20 ml solution. One package may contain oneor more unit doses. Especially preservative-free solutions are oftenformulated in non-resealable containers containing up to about twelve,preferably up to about five units doses, where a typical unit dose isfrom one to about eight drops, preferably one to about three drops. Thevolume of one drop is about 20-40 μL.

6. BIOLOGICAL ACTIVITY

Murine Mixed Lymphocyte Response (MLR) assay: This assay monitoredT-cell proliferation by measurement of tritiated thymidine incorporationinto DNA of T-cells isolated from mouse spleens; T-cell proliferation isdirectly proportional to tritium incorporation into DNA. Pooled spleencells were prepared from three mice each of two strains: three C57BL/6and three BALB/c mice. The C57BL/6 mice were used as the Responders (R)and BALB mice were the Stimulators (S) in the MLR. Complete mediumconsisted of RPMI 1640 supplemented with 25 mM HEPES, 10%heat-inactivated low background fetal bovine serum, 50 μM2-mercatoethanol, 2 mM L-glutamine, and antibiotics.

The BALB/c spleen cells were gamma irradiated at 3000 Roentgen in aBevill irradiator and washed once with medium to use as S cells; thestock of S cells was 1.6×10⁶/mL in complete medium. The R cells werestocked at 8×10⁵/mL in complete medium. Final cell densities were 2×10⁵R and 4×10⁵ S per test well in a 96-well microtiter plate. Compoundswere diluted from a 10 mM stock solution in DMSO into culture medium;compounds were tested at 10 concentrations between 0.001 and 30 μM(three-fold dilutions).

Background control wells contained 150 μL of medium/0.3% DMSO and 50 μLof R cells (2×10⁵/well). All other wells contained 100 μL of medium/0.3%DMSO or compound (also in 0.3% DMSO), 50 μL of R cells (2×10⁵/well), and50 μL of S cells (4×10⁵/well). Plates were incubated at 37° C. in 5% CO₂for 5 days, and subsequently pulsed with 1 μCi/well of ³H-thymidine for6-18 hours. Cells were harvested, and tritium incorporation was measuredusing a scintillation counter.

Inhibition of T-Lymphocyte Activation In Vitro.

Lymphocyte infiltration is one of the main mechanisms of dry eye anduveitis. Therefore, inhibition of T-cell activation provides a basis fortreatment of either disease. Assays with human cells are used todetermine if compounds 1) inhibit T-cell activation (using cytokinerelease as a biomarker), and 2) reduce or prevent cytokine-inducedpathologies.

Compounds were incubated with stimulated human T-lymphocytes in an invitro system designed to measure cytokine production; cytokineproduction is indicative of T-cell activation and consequentialcytokine-induced damage in vivo. Human T-lymphocytes were isolated(Yssel et al, J. Immunol. Meth., 74: 219) from sites of inflammationfrom two human donors, labeled P23 and P26. Cells were treated intriplicate with compounds or vehicle (DMSO) in cell culture medium for 2hours at 37 deg C. Cells were then stimulated with 5 μL ofanti-CD3/anti-CD28 ExpandBeads (DynaBeads, Invitrogen 111.31) andincubated for 24 hours. Compounds were tested at three concentrations:0.02, 0.2 and 2 μM. Untreated controls were performed for each cytokinemarker. The following cytokines were quantitatively determined usingspecific ELISA kits, according to manufacturer instructions: IL2(Eli-pair, Diaclone), IL4 (ImmunoTools 313.300.49), tumor necrosisfactor alpha (TNFα, ImmunoTools 313.33.0.1), and Interferon gamma(IFN-γ) Eli-pair, Diaclone).

TABLE 1 Inhibition of cytokine production in human T-lymphocytes and theMurine-mixed Lymphocyte Reaction (MLR). Approximate IC₅₀ (μM) forcytokine production* IC₅₀ (μM) Cmpd TNF-α IL4 IL2 IFN-γ MLR A 2 2 0.02 20.2 D 2 2 1 1 0.2 F 0.2 0.2 0.02 0.2 0.5 *Average value from results oftests from cultures of two donors.

The compounds listed in Table 1 above inhibited cytokine production fromstimulated human lymphocytes and in the Murine MLR; no cytotoxicity wasobserved at the any concentration tested.

The solubilities of compounds in artificial tear formulations weredetermined using turbidimetric titration (Schote, et al., 2002. J PharmSciences 91(3):856). The different tear formulations are buffered andcontain various physiological electrolytes including, sodium, potassium,phosphate, and chloride. The formulations differ primarily in the“active” ingredients that provide lubrication and viscosity. Compoundswere tested for solubility in various artificial tear formulationscontaining the main classes of active agents: 0.5%carboxymethylcellulose, 0.95% propylene glycol, 0.5% sodiumcarboxymethylcellulose, 1% Polyethylene glycol 400, plus 1% Polyvinylalcohol, and 0.1% Dextran 70 and 0.3% hydroxypropyl methylcellulose. Thesolubilities of N-substituted analogs were determined to be 100 to 2000μM in various artificial tears (see example below).

TABLE 2 Solubility of Compound A in Artificial Tear Formulations:Minimum Other Ingredients pH Solubility (μM) (%) 6.8 130Carboxymethylcellulose sodium (0.5) 7.3 117 Propylene Glycol (0.95),Glycerin (0.3) 6.2 151 Polyethylene glycol 400 (1), Polyvinyl alcohol(1) 7.4 141 Dextran 70 (0.1), Hydroxypropyl methyl-cellulose 2910 (0.3)6.7** 350 Dextran 70 (0.1), Hydroxypropyl methyl-cellulose 2910 (0.3)**pH was adjusted with HCl from 7.4 to 6.7

Evaluation of Ocular Irritancy Potential Using the HET-CAM Test

The compounds of the invention were evaluated for their potential to benon-irritant to the eye in an in vitro test that is accepted to bepredictive of this property known as the HET-CAM test (Steiling, et al.,1999. Toxicology in Vitro 13 (2): 375). In this test, chorio-allantoicmembranes (CAM) of fertilized chicken eggs are exposed to a solution ofthe test substance in sterile water. During an incubation period ofapproximately 5 minutes, irritancy is assessed by visual inspection ofthe CAM blood vessel network for haemorrhage, lysis and/or coagulation.The time of onset or appearance of each of these effects is scorednumerically, the scores are summed to give a single numerical value, andan average numerical score across several eggs is obtained. Based oncomparison to historical control substances and vehicle, aclassification of irritation potential is obtained (Luepke, 1985. FoodChem. Toxicol. 23: 287). The irritancy potential of compounds of thisinvention is summarized below:

TABLE 3 Irritancy Potential Using the HET-CAM Test: Test Total substanceLysis Haemorhage Coagulation Score Classification Vehicle* 3 0 0 3.0Slightly irritant A 3.5 0 0 3.5 Slightly irritant D 3 0 0 3.0 Slightlyirritant F 3 0 0 3.0 Slightly irritant J 3 0 0 3.0 Slightly irritant *1%(v/v) DMSO in sterile water.

Evaluation of Ocular Tolerability in Rabbits.

The compounds of the invention were evaluated for tolerability uponinstillation in the eyes of rabbits over a three day test period.Rabbits receive five instillations within 20 minutes of fiftymicroliters of a solution of the test substance in vehicle once on Day1, twice on Day 2 and four times on Day 3 in the right eyes. Evaluationof ocular irritancy was performed using the Draize scale (Draize et al.J. Pharmacol. Exp. Ther. 1944. 82: 377). In this evaluation, Compound A(0.05% (w/w) in an artificial tear formulation) was very well tolerated,eliciting only slight conjuctival redness not considered to besignificantly different from a vehicle-only treatment group.

The compounds of the invention exhibit both solubility andanti-inflammatory properties that are beneficial in the topicaltreatment of dry eye and uveitis

LPS Induced Uveitis in the Rabbit.

A study is conducted on 12 rabbits to evaluate the effects of thecompound on endotoxin-induced acute uveitis in rabbits, based on theprocedure described by Allen J B et al., Exp. Eye Res. 1996 January;62(1):21-8. To induce acute anterior uveitis, Salmonella typhimuriumlipopolysaccharide endotoxin (LPS) is intravitreally injected into theright eyes of the rabbits. Topical treatment to both eyes of thecompound (right eye) or control (left eye), once every 6 hours, of anoptimal dose, for example 0.1% w/v, is given to 6 rabbits immediatelyfollowing intravitreal injection of 10 ng LPS or vehicle (see tablebelow). The four eye groups include a negative control receiving onlyvehicle (Group 1), a positive-uveitis control without drug treatment(group 2), uveitis eye with drug-treatment (group 3), and each eye istreated and followed for 7 days. Eyes are evaluated for clinicalirritation scores (microscopic ophthalmic examinations), fluorescein dyetest, electroretinography (electrodiagnostic method of retinaltoxicity), aqueous humor protein concentration and cell counts, andcomplete ocular histopathology. After the seven days, a favourableresponse is finding no clinically-significant difference between groups1 and 3.

Testing Topical Treatment for Dry Eye Using Canine KeratoconjunctivitisSicca (KCS) as a Model.

Typical clinical signs of canine KCS include progressive conjunctivitis,superficial keratitis, mucoid ocular discharge, blepharospasm, andultimately corneal scarring and pigmentary keratitis that results inloss of vision. The disease is bilateral and nearly symmetrical.Diagnosis of KCS is made based on the presence of typical clinicalfeatures and a Schirmer tear test (STT) value of less that 10 mm ofwetting/minute (Normal is 15-30 mm/min).

Dogs diagnosed with chronic (>3 months in duration) immune-mediated KCSby a veterinary ophthalmologist are selected for treatment. The study isan open label, single group efficacy study using 50 μl (1 drop) of 0.1%compound in each affected eye twice a day. The efficacy of the drug isevaluated based tear production (as measured by the STT), the responseof clinical observation of the cornea, and the dog owners' andparticipating ophthalmologists' overall assessment of efficacy. Physicaland ophthalmic examinations (biomicroscopy, indirect ophthalmoscopy) areperformed every 2 weeks for the duration of the trial (12 weeks). Theseexaminations include performing STT and assessing severity of the ocularinflammation. The amount of corneal inflammation is subjectively scoredfor each dog during each examination via biomicroscopy, both before andduring the clinical trial. Scores ranging from 0 to 4 (0=normal; 4=mostsevere) are recorded for 3 parameters: area of corneal vascularization(0, 1-25%, 26-50%, 51-75%, and >75%); severity of loss of transparency(conical cloudiness—normal, slight, moderate, severe loss oftransparency, and opaque), and area of corneal cloudiness (0, 1-25%,26-50%, 51-75%, and >75%). Results are substantiated by statisticalanalysis using ANOVA with Tukey-Kramer multiple comparison procedure, aKruskal-Wallis test, a student's t test, and/or calculation of aPearson's correlation coefficient (r). All results and probabilities aregenerated by computerized statistical software (SAS, Inc, Cary, N.C.)and values of P<0.05 are considered significant.

A favorable response to the compound is observed as an increase of ≧5mm/min on the STT and substantial improvement in clinical signs (e.g.,decreased mucus discharge, blepharospasm, conjunctival hyperemia, etc),as observed by the ophthalmologist within 6 weeks.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. While the invention has been described interms of various preferred embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions, andchanges may be made without departing from the spirit thereof.Accordingly, it is intended that the scope of the claimed subject matterbe limited solely by the scope of the following claims, includingequivalents thereof.

1. A method for treating or preventing an ocular disease comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of general formula (I):

wherein: Ak is alkylene; R¹ is —NR⁵R⁶, in which R⁵ and R⁶ are eachindependently hydrogen or straight- or branched-chain alkyl comprisingfrom one to six carbon atoms; or R⁵ and R⁶, together with the nitrogenatom to which they are attached, form a saturated or unsaturatedheterocyclic ring containing from four to six ring atoms, which ring mayoptionally contain another heteroatom selected from the group consistingof nitrogen, oxygen and sulfur and may be optionally substituted by fromone to four groups which may be the same or different selected fromalkyl, hydroxyl, amino, N-alkylamino and N,N-dialkylamino; R² isisobutyl; R³ is (E)-2-butenyl-1 or n-butyl; R⁴ is ethyl, 1-hydroxyethyl,isopropyl or n-propyl; or a pharmaceutically acceptable salt or solvatethereof.
 2. The method of claim 1, wherein R⁵ and R⁶ are eachindependently hydrogen or straight- or branched-chain alkyl comprisingfrom one to six carbon atoms; or R⁵ and R⁶, together with the nitrogenatom to which they are attached, form a saturated or unsaturatedheterocyclic ring containing from five to six ring atoms, which ring mayoptionally contain another heteroatom selected from the group consistingof nitrogen, oxygen and sulfur and may be optionally substituted by fromone to four alkyl groups which may be the same or different.
 3. Themethod of claim 1, wherein Ak is —(CH₂)_(p)—, where p is 1, 2, 3 or 4.4. The method of claim 1, wherein R³ is (E)-2-butenyl-1 and R⁴ is ethyl.5. The method of any one of claims 1 to 4, wherein R¹ is N-morpholine orN,N-dimethylamino.
 6. The method of claim 1 wherein Ak is (CH₂)_(p) andp is two or three; R¹ is —NR⁵R⁶; R⁵ and R⁶, which may be the same ordifferent, are hydrogen or C₁₋₃ alkyl; or R⁵ and R⁶, together with thenitrogen atom to which they are attached, form a saturated orunsaturated heterocyclic ring containing from four to six ring atoms,which ring may optionally contain another heteroatom selected from thegroup consisting of nitrogen and oxygen and may be optionallysubstituted by one or two groups which may be the same or differentselected from methyl, hydroxyl and dimethylamino; R² is isobutyl; R³ is(E)-2-butenyl-1; and R⁴ is ethyl.
 7. The method of claim 1, wherein thecompound of general formula (I) is3-[2-(N,N-dimethylamino)ethoxy]cyclosporin;3-[2-(azetidin-1-yl)ethoxy]cyclosporin;3-[2-(pyrrolidin-1-yl)ethoxy]cyclosporin;3-[2-(piperidin-1-yl)ethoxy]cyclosporin;3-[2-(4-methylpiperazin-1-yl)ethoxy]cyclosporin;3-[2-(morpholin-4-yl)ethoxy]cyclosporin;3-[2-(2,6-dimethylmorpholin-4-yl)ethoxy]cyclosporin;3[2-(3-hydroxypyrrolidin-1-yl)ethoxy]cyclosporin;3-{2-[(4-dimethylamino)piperidin-1-yl]ethoxy}cyclosporin;3-[2-(4-hydroxypiperidin-1-yl)ethoxy]cyclosporin;3-[2-(imidazol-1-yl)ethoxy]cyclosporin;3-[2-(N-methylamino)ethoxy]cyclosporin;3-[2-(N-isopropyl-N-methylamino)ethoxy]cyclosporin; or3-[2-(aminopropoxy)]cyclosporin; or a pharmaceutically acceptable saltor solvate thereof.
 8. The method of claim 1, wherein the disease isselected from the group consisting of dry eye, uveitis andphacoanaphylactic endophthalmitis.
 9. (canceled)
 10. (canceled)
 11. Amethod for treating or preventing an ocular disease comprisingadministering to a subject in need thereof (a) a therapeuticallyeffective amount of a compound of general formula (I) as defined inclaim 1, or a pharmaceutically acceptable salt or solvate thereof; and(b) a second agent effective for the treatment or prevention of theocular disease.
 12. A pharmaceutically acceptable salt of a compound offormula (I) as defined in claim 1.